The invention relates to cancer therapies, to gene therapies for treating cancer, and to in vivo methods for transferring molecules from engineered cells into other cells.
A gap junction is a membrane structure detectable at points of contact between adjacent cells. Gap junctions mediate the intercellular passage of small molecules from the cytoplasm of one cell to that of the adjacent cell.
Gap junctions are composed of clusters of membrane proteins which form structures called connexons. The proteins are peripherally disposed around a central channel. Gap junction transmembrane passages are formed when a connexon of one cell aligns with a connexon of an adjacent cell. In this way, transmembrane intercellular pathways are formed that permit the passage of molecules between coupled cells.
The diameter of the connexon channels, as measured by electron microscopy and X-ray crystallography, is about 1.5-2 nm. Based on this measurement, one would expect that only substances of less than approximately 1,000 daltons (ions, small molecules and synthetic oligonucleotides, but not macromolecules such as proteins and polynucleotides) would enter the channel and pass from one cell to another. This prediction has been verified experimentally. Sugars, nucleic acids, amino acids, fatty acids, small peptides, and even drugs or carcinogens could pass between cells through gap junctions. Proteins, complex lipids, polysaccharides, RNA, and other large molecules do not. This intercellular channel flux of ions and molecules does not require energy and is reportedly due to passive diffusion.
The protein subunits of connexons may vary from cell to cell. Some connexons contain ductin. Finbow and Pitts, J. Cell Sci. 106: 463-472 (1993); Leitch and Finbow, Exp. Cell Res. 190: 218-226 (1990); Bohrmann, J. Cell Sci. 105: 513-518 (1993). Some connexons contain connexins. See Beyer et al., J. Membr. Biol., 116:187-194 (1990), and references cited therein.
More than a dozen connexon proteins have been cloned from different species. The best characterized of the connexon proteins, the connexins, are the gene products of a multi-gene family. Connexins are expressed in a cell-, tissue-, and developmentally specific manner. See Beyer et al., J. Membr. Biol., 116:187-194 (1990); Dermietzel, R. et al., Anat. Embryol., 182:517-258 (1990; Warner, A., Seminars in Cell Biology, 3:81-91 (1992); Kumar, N. M. et al., Seminars in Cell Biology, 3:3-16 (1992). For instance, connexin43 is the predominant connexin expressed in cardiac muscle and in liver epithelial cells. In adult liver parenchymal cells, the predominant connexins are connexins32 and connexin26; nonparenchymal liver cells express other connexins. Each connexin forms channels with different conductance, regulatory, and permeability properties.
In those tissues where more than one connexin is expressed, gap junctions may contain more than one connexin. However, it is not known whether individual connexons may be comprised of more than one connexin type.
Individual cells that are coupled by gap junctions to neighboring cells do not always behave as discrete entities. Rather, two or more coupled cells often function in a highly integrated manner. Instead of responding individually to certain environmental stimuli, they respond as a unit. In this way tissues can buffer or eliminate toxic compounds that would otherwise be harmful to individual cells, healthy cells can nourish injured cells, and critical molecules can be distributed among cells to coordinate local functions. For example, it has been proposed that gap junctions play an important role in the function of cardiac muscle cells. The rapid intercellular exchange of ions through gap junctions is believed to mediate coordinated contraction of the myocardium.
Gap junctional communication appears to contribute to what has been variously termed the "kiss of death," the "kiss of life" or the "bystander effect". When cells that are normally insensitive to a given substance are connected via gap junctions to cells that are normally sensitive, the insensitive cells become sensitive ("kiss of death"), reportedly because of the passage of toxic molecules from the sensitive to the insensitive cells. On the other hand, when certain cells that are unable to survive in defined tissue culture media are joined to cells that are capable of surviving, in some instances the sensitive cells acquire the ability to survive, reportedly because of the passage of molecules from survivalist cells to sensitive cells ("kiss of life").
The ability of adjacent cells to form gap junctions that link them is dependent on a number of factors:
a) the ability of cells to interact with neighboring cells. It has been reported that the presence of compatible cell adhesion molecules is an important requirement of gap junctional formation between adjacent cells. See Edelman, G. M., Biochem, 27:3533-3543 (1988); Meyer, R. A. et al., J. Cell Biol., 119:179-189 (1992); Keane, R. W. et al., J. Cell Biol., 106:1307-1319 (1988); Musil, L. S. et al., J. Cell Biol., 111:2077-2087 (1990); Jongen et al., J. Cell Biol., 114:545-555 (1991); Hofer et al. J. Neuroscience 16(14)4311-21 (1996). PA1 b) it has been reported that increased levels of connexon protein expression results in increased gap junction communication. See Eghabali et al., Proc. Natl. Acad. Sci. USA, 87:1328-1331 (1990); Zhu et al., Proc. Natl. Acad. Sci. USA, 89:10218-10221 (1992); and Zhu et al., Proc. Natl. Acad. Sci. USA, 88:1883-1887 (1991); Mesnil et al. Proc. Natl. Acad. Sci. USA, 93(5):1831-5.(1996); Bruzzone et al. Eur. J. Biochem. 238(1):1-27 (1996); Shinoura et al., J. Neurosurg. 84(5):839-45 (1996); Fishman et al., Cell Adhesion and Communication 3(4):353-65 (1995); Elfgang et al. J. Cell Biol. 129(3):805-17 (1995); Koval et al., J. Cell Biol. 130(4):987-95 (1995); Moore et al., Amer. J. Physiol. 267(5 Pt 1):C1371-80 (1994); PA1 c) whether the connexon proteins expressed by one cell are capable of forming a connexon that is capable of linking with a connexon of a second cell to form a functional channel. PA1 d) whether a molecule such as a carcinogen or the product of an oncogene is expressed in a cell, that interferes with the normal function of any cellular protein that mediates cell to cell gap junctional communication. See Holder et al., Cancer Research, 53:3475-3485 (1993). PA1 a) providing engineered non-tumorigenic cells that express a first heterologous nucleic acid that encodes a molecule which enables, mediates or enhances the gap junctional transport of therapeutic molecules and a second heterologous nucleic acid which encodes a molecule with desired biological effects capable of passing through a gap junction or a pro-drug activating molecule that converts a non-toxic substrate to a toxic metabolite. PA1 b) contacting tumor cells in a solid tumor with cells of step a that form functional gap junctions with said tumor cells, and then PA1 c) in the case of therapeutic strategies utilizing pro-drug activating molecules, exposing the engineered and tumor cells to the non-toxic substrate, whereby the non-toxic substrate is converted to the toxic metabolite in cells that expresses the pro-drug activating molecule, and the toxic metabolite then passes through gap junctions to adjacent coupled tumor cells, thereby inhibiting the cell growth of the tumor cells that contain the toxic metabolite. PA1 a) providing engineered cells that contain or can generate a therapeutically active molecule that passes through gap junctional channels, wherein the engineered cells express a heterologous nucleic acid that encodes a molecule that enables or enhances the extent of gap junction mediated intercellular communication. Such molecules include but are not limited to cellular adhesion molecules or connexon proteins that are expressed by the tumor cells described in step b, and PA1 b) contacting target tumor cells in a solid tumor with cells of step a that form functional gap junctions with said target tumor cells, whereby the molecule passes through a gap junction to a target tumor cell.
It has been reported that cancer cells exhibit reductions in gap junction-mediated intercellular communication compared to normal cells with a similar tissue origin. While neoplastic cells can sometimes form gap junctions with other types of cells (heterologous coupling), it is recognized that gap function formation between cells of a similar type (homologous coupling) can be more efficient.
It has been repeatedly observed that coupling normal cells to tumor cells slows down the growth of tumor cells. Also, experimental techniques that result in an increase in gap function channels can reduce cellular proliferation and tumorigenicity.
Since gap junctional communication appears to play a role in neoplasia, it has been suggested that increasing gap junctional communication should reverse neoplasia. Connexin gene expression can be increased in malignant cell lines by the introduction of connexin genes. The proliferation rate of tumor cell growth in tissue culture, as well as tumor formation after injection into animals have been found to be reduced in cells that were genetically engineered to overexpress connexin genes.
A new method for tumor gene therapy uses recombinant retroviral vectors containing a gene (HSVtk) that encodes the thymidine kinase enzyme of the herpes simplex virus (HSVTK). Following infection of a tumor cell by the retroviral vector, the HSVtk gene is expressed therein. The HSVTK enzyme avidly phosphorylates certain nucleoside analogues, such as the drug Ganciclovir (GCV), for which normal mammalian thymidine kinase has a poor affinity. These phosphorylated analogues are incorporated into the cellular genome during DNA replication, leading to tumor cell death. Exposure to GCV does not appear to harm normal cells when given in therapeutic doses.
A recognized limitation of this therapy is the relative inefficiency of gene transfer. Also, retroviral particles are relatively rapidly inactivated by host organism defenses, such as complement. In addition, a target cell must be replicating for the DNA to be stably integrated. Only a minority of tumor cells can be genetically altered after retroviral infection, especially in vivo.